Identificação de variantes somaclonais em bananeiras 'Prata Anã', utilizando técnicas moleculares e citogenéticas.

Variação somaclonal é uma variação fenotípica de origem genética, ou seja, uma variação cromossômica que se torna herdável nas gerações seguintes, ou epigenética, que é uma variação transitória devido ao estresse fisiológico que o material sofre, quando submetido ao cultivo in vitro. Um problema específico envolvendo a variação somaclonal em bananeiras 'Prata Anã' foi observado em Andradas, Minas Gerais, em plantas oriundas de micropropagação. A maior dificuldade na separação dos indivíduos normais e variantes é que os caracteres morfológicos, que são inerentes a este tipo de variação, só se tornam evidentes quando a planta está adulta, o que impossibilita a eliminação dos indivíduos variantes ainda em viveiro. Com o objetivo de identificar, ainda em viveiro aqueles indivíduos variantes somaclonais, técnicas moleculares (RAPD e SSR) e citogenéticas (contagem cromossômica e citometria de fluxo) foram utilizadas. Cento e três primers RAPD, 11 combinações de dois primers RAPD, e 33 pares de primers SSR foram utilizados na tentativa de se encontrar marcadores polimórficos capazes de distinguir os indivíduos normais dos variantes, além de distinguir bananeiras 'Prata Anã' de 'Prata'. O primer OPW-08 gerou um fragmento polimórfico que distinguiu uma planta variante de todas as demais, provando que a variação não ocorre de maneira uniforme no genoma dos indivíduos variantes e que não há um retorno à cultivar Prata. As análises com marcadores SSR e a contagem cromossômica não possibilitaram a distinção dos indivíduos variantes, nem a separação das cultivares Prata e Prata Anã. As análises de citometria de fluxo evidenciaram a grande instabilidade cromossômica das bananeiras, porém elas não foram eficientes na identificação de variantes somaclonais

ASYMMETRIC LEAVES2-LIKE1gene a member of the AS2/LOB family, controls proximal-distal patterning in Arabidopsis petals

The formation and the development of the floral organs require an intercalate expression of organ-specific genes. At the same time, meristem-specific genes are repressed to complete the differentiation of the organs in the floral whorls. In an Arabidopsis activation tagging population, a mutant affected in inflorescence architecture was identified. This gain-of-function mutant, designateddownwards siliques1 (dsl1-D), has shorter internodes and the lateral organs such as flowers are bending downwards, similar to the loss-of-function brevipedicellus (bp) mutant. The affected gene in dsl1-D appeared to be ASYMMETRIC LEAVES2-LIKE1 (ASL1)/LATERAL ORGAN BOUNDARIESdomain gene 36 (LBD36), which is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. Analysis of the loss-of-function mutant asl1/lbd36 did not show morphological aberration. Double mutant analysis of asl1/lbd36 together with as2, the ASL1/LBD36 closest homologue, demonstrates that these two members of the AS2/LOB family act partially redundant to control cell fate determination in Arabidopsis petals. Moreover, molecular analysis revealed that overexpression of ASL1/LBD36 leads to repression of the homeobox gene BP, which supports the model that an antagonistic relationship between ASL/LBD and homeobox members is required for the differentiation of lateral organ

Effect of pruning strategy on 'Syrah' bud necrosis and fruitfulness in Brazilian subtropical Southeast

The change of wine grape harvest from wet season (summer) to dry season (winter) by changing the pruning management has improved quality of wines produced in the Brazilian Southeast. However, the vines need to be spur pruned twice a year, i.e. with a 1st pruning in August (winter pruning) for a vegetative cycle during the hot and wet summer, and a 2nd pruning in January (summer pruning) for a productive cycle during the cold and dry season. This double pruning strategy is made necessary by the fact that latent buds developed during the dry season cycle are not fruitful to support a productive cycle in the following year. This histological study, performed in the South of Minas Gerais State (Brazil), showed that annual single pruning done in the wet season (in January) displayed a high rate of necrosis on primary and secondary buds (bud necrosis – BN). In April, 99 days after summer pruning (DASP), the rates of BN were 40 % and 50 % at basal and apical node positions, respectively, reaching 80 % of BN in December (322 DASP). As a consequence of BN, bud potential fertility was drastically reduced from 0.5 inflorescence primordial (IP) per bud (in July) to 0.06 (in December) and bud burst in the next cycle from secondary and tertiary bud axes. Vines managed by double pruning system (submitted to summer and winter pruning) displayed a much higher fruitfulness potential, i.e. 1.46 IP per bud in December (112 days after winter pruning) and limited BN occurrence (20 %). On single pruned vines, we also observed a significant decrease of starch content in canes, trunks and roots. Internal bud anatomy showed that a random cell breakdown started 70 days DASP. At 211 DASP, all buds showed a large starch granule concentration, raphides and crystals of calcium oxalate inside idioblasts of leaf primordia and also in cortical parenchyma of the vegetative axis. The bud starch content was increased and a positive correlation between necrosis and starch accumulation was observed. The impact of carbohydrate availability on bud necrosis development was discussed. This study showed that the necrosis development towards secondary and tertiary axis of the dry season buds is the main reason of unfruitfulness in the vineyards managed by single pruning in the wet season, making the double pruning compulsory

Regulation of Plant Developmental Processes by a Novel Splicing Factor

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes

Molecular analysis of plant architecture in Arabidopsis thaliana using activation tagging.

Keywords: Arabidopsisthaliana, activation tagging, T-DNA, transposon, mutants, enhancer, DNA methylation, plant architecture, development, forward/reverse genetics, lateral organs, flower, vascular tissue, HLH, transmembrane, transcription factorsPlant development is one of the most important aspects of plant's life cycle that has extensively been studied at the morphological, genetic and molecular level. It is import for systematic and taxonomic classification, but also for applied agronomic reasons, because it affects the growth and cultivation leading to higher yield and quality of the product.The generation of genetic variants, like mutants may increase genetic pool and gives information about plant processes and their genetic control.Activation tagging is a new powerful tool to generate and identify new mutants, which emerged as an alternative for gene function analysis. This thesis reports the study on the molecular control of plant architecture, using mutants generated by an activation tagging-based approach in the model plant Arabidopsis thaliana . In addition, it also describes experiments that could explain why the low frequencies of mutants were obtained by T-DNA based activation tagging. Based on this comparison, the transposon-based activation tagging strategy was chosen and a screen for flower and silique mutants in a large Arabidopsis population yielded three gain-of-function mutants. These mutants were designated downwards siliques1 ( ds1-D ), needle1 ( ndl1-D ) and twisted1 ( twt1-D ). In the ds1-D mutant, internodes are shorter and the lateral organs such as flowers are bending downwards. Further molecular and genetic studies on this mutant revealed that DS1 is important to control petiole-blade boundary in Arabidopsis petals. In the ndl1-D mutant, the normal formation of valve tissues is altered, resulting in a pin-like structure that replaces the two fused carpels of the wild type pistil. The results suggest that NDL1 is involved in normal carpel development, in which auxin distribution plays an important role. In the third mutant, twt1-D , the overexpression of TWT1 led to twisting of all organs, whichismost pronounced in siliques. This phenotype and the expression pattern of the gene suggest that TWT1 is involved in proper vascular tissue development in Arabidopsis . These studies demonstrate the power of activation tagging and it gains valuable knowledge about the molecular networks that control plant development

Analysis of the SHP2 enhancer for the use of tissue specific activation tagging in Arabidopsis thaliana

Activation tagging is a powerful tool to identify new mutants and to obtain information about possible biological functions of the overexpressed genes. The quadruple cauliflower mosaic virus (CaMV) 35S enhancer fragment is a strong enhancer, which is most commonly used for this purpose. However, the constitutive nature of this enhancer may generate lethal mutations or aberrations in different plant organs by the same overexpressed gene. A tissue-specific activation tagging approach may overcome these drawbacks and may also lead more efficiently to the desired phenotype. For this reason the SHATTERPROOF2 (SHP2) promoter fragment was analysed for enhancer activity. The SHP2 gene is involved in dehiscence zone development and expressed during silique development. The aim of the experiments described here was to identify a dehiscence zone specific enhancer that could be used for tissue-specific activation tagging. The chosen SHP2 enhancer fragment was found to be expressed predominantly in the dehiscence zone and showed enhancer activity as well as ectopic expression activity. This activity was not influenced by its orientation towards the promoter and it was still functional at the largest tested distance of 2.0 kb. Based on these results, the SHP2 enhancer fragment can potentially be used in a tissue-specific activation tagging approach to identify new Arabidopsis mutants with an altered dehiscence zone formation

Low frequency of T-DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences

A powerful system to create gain-of-function mutants in plants is activation tagging using T-DNA based vehicles to introduce transcriptional enhancer sequences. Large Arabidopsis populations of individual plants carrying a quadruple cauliflower mosaic virus (CaMV) 35S enhancer are frequently used for mutant screenings, however the frequency of morphological mutants remains very low. To clarify this low frequency we analyzed a subset of lines generated by this method. The correlation between the number of T-DNA insertion sites, the methylation status of the 35S enhancer sequence and 35S enhancer activity was determined. All plants containing more than a single T-DNA insertion showed methylation of the 35S enhancer and revealed a dramatic decrease in 35S enhancer activity. The results support the notion that in a large proportion of the T-DNA based activation tagged lines the 35S transcriptional enhancer is silenced due to methylation, which is induced by multiple T-DNA integrations